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Rapid visual genotyping method for germline mutants with small genomic fragment deletion by allele-specific PCR and lateral flow nucleic acid biosensor

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Abstract

Background

Genome-editing techniques incorporating artificial nucleases develop rapidly and enable efficient and precise modification of genomic DNA of numerous organisms. The present research aimed to establish a rapid, sensitive and visual method for genotyping of germline genome-edited mutants with small genomic fragment deletion.

Methods and Results

The genome-edited pigs with 2-bp deletion and 11-bp deletion of Myostatin (MSTN) gene generated by TALENs system were used as test materials to check the proposed allele-specific PCR (AS-PCR) and lateral flow nucleic acid biosensor (LFNAB) cascade method. AS-PCR can produce products with different tags to distinguish genome-edited alleles and wild-type alleles. A LFNAB was applied to do visual detection of AS-PCR products without using additional instruments. Furthermore, we demonstrated that AS-PCR and LFNAB cascade could accurately and visually distinguish genome-edited pigs with small genomic fragment deletion of Myostatin (MSTN) gene and wild-type pigs with limit of detection (LOD) of 0.1 ng.

Conclusion

The proposed AS-PCR and LFNAB cascade can do rapid and visual genotyping of genome-edited mutants with small genomic fragment deletion, serving as a platform for genome-edited animal genotyping.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (31802040 and 32172699).

Author information

Author notes

  1. Qiuju Su and Xiang Zhou have contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, 430070, China

    Qiuju Su, Xiang Zhou & Bang Liu

  2. The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, China

    Xiang Zhou & Bang Liu

  3. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China

    Tianwen Wu & Kui Li

  4. Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, 100083, Beijing, China

    Wentao Xu

  5. MOE Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou, 350002, Fujian, China

    Zhenyu Lin

  6. Development Center for Science and Technology, Ministry of Agriculture and Rural Affairs, Beijing, 100045, China

    Ping Shen

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  1. Qiuju Su
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  8. Bang Liu
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Contributions

QS, XZ and BL conceived of the AS-PCR and LFNAB cascade method. QS performed the experiments. TW and KL contributed materials. QS and XZ wrote the manuscript. XZ, LB, WX, ZL, and PS edited the manuscript.

Corresponding author

Correspondence to Bang Liu.

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Su, Q., Zhou, X., Wu, T. et al. Rapid visual genotyping method for germline mutants with small genomic fragment deletion by allele-specific PCR and lateral flow nucleic acid biosensor. Mol Biol Rep 48, 7325–7332 (2021). https://doi.org/10.1007/s11033-021-06734-x

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